Cell and tissue culture container

ABSTRACT

The invention generally relates to containers for cell and tissue culturing with multiple compartments in fluid communication with each other to provide a common culture environment in each of the compartments while maintaining physical separation of cells and tissue therein. The invention further relates to culture containers providing a sterile culture environment with detachably coupleable lids and open access to each compartment within a container.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/558,080, filed Dec. 2, 2014, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to cell and tissue culture devices.

BACKGROUND

While certain tissues in the human body, such as skin, are capable ofself-repair (e.g., wound healing), there are many tissues that are not.For example, articular cartilage has no innate ability to repair itself,rendering any damage thereto permanent. Articular cartilage linesopposing bone surfaces in diarthrodial joints and provides a smooth,lubricated surface for articulation. Accordingly, defects in articularcartilage tend to expand and worsen over time. Damage to the articularcartilage in joints such as the knee can lead to debilitating pain.

Typical treatment choices, depending on lesion and symptom severity, arerest and other conservative treatments, minor arthroscopic surgery toclean up and smooth the surface of the damaged cartilage area, and othersurgical procedures such as microfracture, drilling, and abrasion. Allof these may provide symptomatic relief, but the benefit is usually onlytemporary, especially if the person's pre-injury activity level ismaintained. For example, severe and chronic forms of knee jointcartilage damage can lead to greater deterioration of the jointcartilage and may eventually lead to a total knee joint replacement.Approximately 200,000 total knee replacement operations are performedannually. The artificial joint generally lasts only 10 to 15 years andthe operation is, therefore, typically not recommended for people underthe age of fifty.

An alternative treatment is implantation of cultured neo-cartilage(i.e., immature hyaline cartilage) which can be grown in-vitro to adesired size and shape on a 3D scaffold from chondrocyte cells biopsiedfrom the patient (autologous) or from another individual (heterologous).Examples of this process are described, for example, in U.S. Pat. Nos.6,949,252; 7,537,780; 7,468,192; 7,217,294; and U.S. patent applicationSer. No. 14/208,931. An exemplary method for 3D culture of neo-cartilageis shown in FIG. 8 and includes the steps of isolating chondrocyte cellsfrom a biopsy, 2D growth of cells, seeding of a 3D scaffold, and twoculturing steps. The first culturing step takes place under controlledpressure, oxygenation, and perfusion conditions to mimic the jointenvironment while the second culturing step is a 3D static culture.

Along with the neo-cartilage for implantation, multiple other surrogatetissues are simultaneously cultured in the same vessel in order topermit pre-implantation testing and verification procedures withoutdamaging the neo-cartilage to be implanted.

Current culture containers include narrow opening flask-type containerswith a sealing cap with a gas-permeable filter membrane but the 3Dstatic culture procedure presents multiple challenges which are unmet bycurrent culture containers. For example, the surrogates and theneo-cartilage to be implanted need to be cultured in the same conditionsin a common fluid to enable validation through surrogate testing.However, in current containers, the surrogates and neo-cartilage cangrow into one another as they mature and then require separation whichcan damage the cells and scaffolds. Another problem stems from the factthat the tissues must be submerged in fluid during the 3D static culturebut the buoyancy of the cultures varies during tissue growth. The 3Dstatic culture process takes 2 weeks and requires incubation throughoutthe process, taking up space in expensive incubators and limitingproduction efficiency and capacity using bulky flask-type containers.Additionally, the surrogates and the neo-cartilage to be implanted mustbe transferred from one container to another during the 2 week processand then must be removed from the container before final packaging fordistribution and implantation. Manipulation of the neo-cartilage to beimplanted and the surrogates through the narrow opening of the currentculture containers is difficult and can cause damage to the cells. Thesechallenges are not unique to 3D static culturing of neo-cartilage andapply to a variety of cell and tissue culturing procedures.

SUMMARY

The invention relates to containers for cell and tissue culturing withmultiple compartments in fluid communication with each other to providea common culture environment in each of the compartments whilemaintaining physical separation of cells and tissue therein. Culturecontainers of the invention can be used to simultaneously culture one ormore surrogate tissues alongside and under the same conditions as atissue to be implanted, thereby enabling destructive testing andverification procedures to be carried out without harming the tissue tobe implanted. Each compartment may contain the same type of tissue ordifferent tissue types in order to investigate interactions betweendifferent tissue types. In some instances multiple tissue types may becultured in a single compartment. The containers of the presentinvention provide containers for culturing these tissues in a commonenvironment and fluid while preventing the surrogate tissues and thetissue for implantation from adhering or growing onto the container oreach other and potentially damaging the tissue for implantation.Containers of the invention may provide sealing lids with gas permeablemembranes to allow for gas exchange between the interior and exteriorenvironments of the container while maintaining a sterile internalenvironment. The culture containers and lids may be configured tominimize height while maintaining adequate interior volume andmaximizing incubator efficiency and space. Containers of the inventionmay also provide multiple compartments in fluid communication yetcapable of restricting movement of and various sizes of sometimesbuoyant tissue cultures and other materials. Additionally, culturecontainers of the invention may provide a large opening with easy,lid-off access to each compartment and any tissue or other materialstherein. The several features of the containers of invention provide amore efficient platform for culturing and verifying cells and tissuesfor implantation while minimizing the potential for damage to theimplantable tissue.

Culture containers of the invention may comprise a bottom wall and atleast one side wall coupled thereto. Side walls may form right angleswith the bottom wall or may taper out to provide a large opening witheasy access to the compartments therein. Culture containers can be acylinder, a cuboid, a triangular prism, a pentagonal prism, an octagonalprism or a variety of other 3 dimensional shapes.

Containers of the invention may include one or more interior walls whichcan divide the interior volume of the container into two or morecompartments. A container may have 11 or more compartments or as few astwo. The compartments may be of the same size or a variety of sizes. Insome instances a larger compartment may be configured to containcultured tissue for implantation while several smaller compartments maybe sized to contain surrogates for testing.

In certain aspects, the container may comprise multiple components suchas a first unit comprising a bottom wall and at least one side wall anda second unit comprising one or more of the interior walls or partitionso that the partition may be removed from the container providing asingle compartment or inserted into the container to provide multiplecompartments therein. In certain instances, a single first componentcomprising the one or more side walls and the bottom wall may becompatible with multiple different second components comprising interiorwalls in various configurations so that, by interchanging secondcomponents, the number of compartments may be varied.

In order to achieve fluid communication between the compartments of thecontainer, the interior walls may have openings including pores, slits,or gaps between an interior wall and a side wall, the bottom wall, orthe lid. The openings may be dimensioned so as to allow fluid to passbetween the compartments of the container without allowing cells,tissues, or 3D scaffolds to pass therebetween. Screens or filters may beused to cover openings in order to help restrict passage of cells,tissues, or 3D scaffolds therethrough.

Devices of the invention may include a lid configured to detachablycouple to the top edge of the one or more side walls in order to enclosethe container. The lid can generally correspond in shape and size to theopening formed by the top edge of the one or more side walls. The lidmay be configured to form an air or water-tight seal when detachablycoupled to the container, may include a gasket to aid in sealing, andmay be secured via complementary threads, interlocking tabs, or othermeans. Preferably, the lid and the container may be detachably coupledwithout the use of tools in order to promote sterility and relativelyeasily in order to avoid excessive motion of the contained fluid anddisruption of the cultured materials.

The lid may comprise a vent and/or a gas permeable membrane of a sizeand shape configured to allow gas to pass into and out of a sealedcontainer while restricting undesirable particles such as bacteria,endotoxins, and other contaminants. The lid may be configured so that,when detachably coupled to the container, the only avenue for gasexchange between the interior and exterior of the container is throughthe filter. A filter may also be located on a side wall of thecontainer.

Containers of the invention may be sized so that containers may containan adequate volume of fluid (e.g., 500 mL) while maintaining sufficientspace between the fluid and the lid to avoid contact during movement ofthe container and so that multiple containers can fit on a standardincubator tray to maximize space efficiency during the incubationprocess.

The lid, the container, or components thereof may comprise a variety ofmaterials including, for example, polypropylene, high-densitypolyethylene, polystyrene, styrene-acrylonitrile (SAN),polycarbonatestyrene-maleic anhydride (SMA), cyclic olefin copolymers(COC), acrylic, acrylic-polyvinyl chloride alloys, polypropylenecopolymers, polysulfone, polymethylpentene, or cellulosic. The lid, thecontainer, or components thereof may comprise a substantiallytransparent material to allow visual inspection of the container'scontents from outside of the container. Containers and lids may beconfigured as single-use disposables or may be configured for repeateduse and sterilization.

In some aspects, the interior surface of the bottom wall, the interiorsurface of the one or more side walls, and/or the surfaces of the one ormore interior walls may be smooth; textured with ridges, valleys, pores,or dimples; and/or treated with an agent to deter adherence of culturedcells or tissues and/or the facilitate retrieval from the container byscooping under the cultured tissue with a retrieval tool.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side view of an exemplary embodiment of a circularmulti-compartment culture container of the invention.

FIG. 2 shows a top view of an exemplary embodiment of a circularmulti-compartment culture container of the invention.

FIG. 3 shows a top view of an exemplary embodiment of a rectangularmulti-compartment culture container of the invention.

FIG. 4 shows a close up view of a single compartment of an exemplaryembodiment of a circular multi-compartment culture container.

FIG. 5A shows a side view of an exemplary embodiment of a lid for acircular culture container.

FIG. 5B shows a top view of an exemplary embodiment of a lid for acircular culture container.

FIGS. 6A and 6B show an exemplary embodiment of a two componentmulti-compartment circular culture container.

FIG. 7 shows an exemplary embodiment of interlocking tabs.

FIG. 8 shows an exemplary method for 3D culture of neo-cartilage forimplantation.

DETAILED DESCRIPTION

The invention relates to culture containers comprising multiplecompartments configured to be in fluid communication with each otherwhile maintaining physical separation of cell or tissues being culturedtherein. Cells and tissues are cultured on 3D scaffolds to form tissuesof a desired shape and size. If a tissue is being cultured forimplantation, it is advantageous to simultaneously culture one or moresurrogate tissues under the same conditions to enable destructivetesting and verification procedures to be carried out without harmingthe tissue to be implanted. The containers of the present inventionprovide containers for culturing these tissues in a common environmentand fluid while preventing the surrogate tissues and the tissue forimplantation from adhering or growing onto the container or each otherand potentially damaging the tissue for implantation. In variousembodiments, each compartment in a container may contain the same typeof tissue or different tissue types in order to investigate effects ofdifferent types of tissue on each other in a shared culture environment.In certain instances multiple tissue types may be cultured in a singlecompartment to investigate physical interactions between the tissues.Culture containers of the invention may be used to culture a variety ofcells and tissues including chondrocytes, mesenchymal stem cells,fibroblasts, osteocytes, osteoblasts, synoviocytes, induced pluripotentstem cells (iPSC), embryonic stem cells (ESC), lymphocytes, adipocytes,myofibroblasts, hepatocytes, islet cells, monocytes, endometrialregenerative cells, or cancer stem cells. Applications for thesecontainers include the culturing of neo-cartilage from chondrocytes forrepair of articular cartilage defects.

Culture containers of the invention may comprise a bottom wall and atleast one side wall coupled thereto. Side walls can be coupled to thebottom wall so that the side wall's planar surface is substantiallytransverse to the planar surface of the bottom wall. The culturecontainer may have any number of side walls including 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 side walls. Side walls may be substantially straight ormay be curved. The bottom wall may be a variety of shapes including,rectangular, circular, triangular, or pentagonal. The bottom wall shapeshould correspond to the number of side walls. For example, a container101 with circular bottom wall 105 would comprise a single side wall 109,with a curved planar surface so that the bottom edge of the single sidewall 109 forms a circle of substantially equal radius to that of thebottom wall 105 to which it is coupled as shown in FIG. 1 .Alternatively, a container may have 4 straight side walls 109 coupled toa substantially rectangular bottom wall 105 as shown in FIG. 3 . Inembodiments having more than one side wall 109, the side walls may becoupled together at a vertical edge to form a corner as shown in FIG. 3.

Containers of the invention may include one or more interior walls whichcan divide the interior volume of the container into two or morecompartments. Interior walls may be curved or straight and can becoupled to the bottom wall so that their planar surface is substantiallytransverse to the planar surface of the bottom wall. Interior walls canbe coupled to the same planar surface of the bottom wall as the sidewalls. Interior walls may be coupled to one or more side walls and/orone or more other interior walls. A culture container may have anynumber of interior walls, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10interior walls and may comprise any number of compartments including 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 compartments.

FIG. 1 illustrates an exemplary embodiment of a circular,multi-compartment culture container 101 having a circular bottom wall105 and a single, curved side wall 109. The container 101 has a singlecurved interior wall 107, coupled to the bottom wall 105 to define acylindrical center compartment 103. The container 101 also has severalstraight interior walls 107 coupled to both the curved side wall 109 andthe curved interior wall 107, forming spokes and defining 10 smallerradial compartments 103 for a total of 11 compartments 103.

FIG. 2 illustrates a birds-eye view of the circular, multi-compartmentculture container 101 of FIG. 1 , having 10 compartments 103 defined bya curved side wall 109, a curved interior wall 107 and 10 straightinterior walls 107.

FIG. 3 shows a birds-eye view of an exemplary container 103 having arectangular bottom wall and 4 side walls 109 defining 8 compartmentsincluding 1 larger compartment 103 and 7 smaller compartments.

The larger center compartment 103 of the containers 101 shown in FIGS.1-3 may be sized to contain a larger piece of tissue for implantationwhile smaller surrogate tissues may be contained in the smallersurrounding compartments 103.

In certain aspects, the container may comprise multiple components suchas a first unit comprising a bottom wall and at least one side wall anda second unit comprising one or more of the interior walls or partitionso that the partition may be removed from the container providing asingle compartment or inserted into the container to provide multiplecompartments therein. One or more of the components may have slots orother physical mechanisms configured to accept a portion of the othercomponent. FIGS. 6A and 6B depict an exemplary embodiment of amulti-component container. A first component 120 is shown in FIG. 6Acomprising a single, curved side wall 109 coupled at its bottom edge toa circular bottom wall 105. FIG. 6B illustrates a second component 130comprising a single, circular interior wall 107 and 10 straight interiorwalls 107 extending radially therefrom. The second component 130 isconfigured to be placed within the first component 120 in order toseparate the interior volume of the first component into 10compartments. The second component 130 may be dimensioned so that theradial edges of the straight side walls 107 form a tight fit with theinner planar surface of the curved side wall 109 when the two componentsare combined.

In certain instances, a single first component comprising the one ormore side walls and the bottom wall may be compatible with multipledifferent second components comprising interior walls in variousconfigurations so that, by interchanging second components, the numberof compartments may be varied.

The first component may include a stop 125 as illustrated in FIG. 6Aconfigured to prevent the bottom edge of the interior walls 107 fromcontacting the interior planar surface of the bottom wall 105.

In order to achieve fluid communication between the compartments of thecontainer, the interior walls may have openings. In certain embodimentsthe openings may be dimensioned so as to allow fluid to pass between thecompartments of the container without allowing cells, tissues, or 3Dscaffolds to pass therebetween. In some aspects an interior wall may beperforated with one or more holes or pores. In such instances, each holeshould have a diameter smaller than the largest dimension of the cell,tissue, or 3D scaffold being cultured within a compartment defined inpart by that interior wall. For example, for 3D tissue culture scaffoldshaving a 4 mm diameter by 1.5 mm thickness, openings in the interiorwalls may have diameters less than 4 mm as the scaffolds should beunable to pass through an opening with a diameter less than 4 mm. Incertain aspects, an interior wall may comprise a slit of a certainlength and width. In such instances, one of the length or the width ofthe slit should be less than the smallest dimension of the cell, tissue,or 3D scaffold being cultured within a compartment defined in part bythat interior wall. For example, using 3D tissue culture scaffoldshaving a 4 mm diameter by 1.5 mm thickness, the length or width of theslit may be any value so long as the other dimension is less than 1.5mm.

Openings may be located at a variety of positions within an interiorwall including near the center, top, or sides of an interior wall.Openings may be uniformly or differentially located on each interiorwall. In certain instances, an opening in an interior wall may belocated near the bottom wall of the container. Cell and tissue culturesmay rest on the bottom wall of the container during the culturingprocess. Openings near the bottom wall can allow for the exchange,between compartments, of fluid, nutrients, growth factors, and otherparticles at the tissue level. Particles that may tend to settle to thebottom may be better exchanged through openings near the bottom wall,thus better replicating the culture environment across compartments.

FIG. 4 illustrates an exemplary embodiment of a compartment 103 within alarger, cylindrical container such as the container 101 illustrated inFIG. 1 . The compartment 103 in FIG. 4 is defined by a portion of acurved side wall 109, a portion of a curved interior wall 107, twostraight interior walls 107, and a portion of a circular bottom wall105. Each of the interior walls 107 has one or more openings 115 nearthe bottom wall enabling the passage of fluid between the compartment103 and other, surrounding compartments (not shown). As noted above, theopenings 115 may be dimensioned to allow fluid to pass through but torestrict the passage of the cultured cell or tissue within thecompartment 103. In certain aspects, openings between compartments, ininterior walls, may be covered by screens or filters configured torestrict inter-compartment passage of cells tissues or 3D scaffolds. Insuch instances the screen or filter may be of a biocompatible materialand configured, by material, conformation, or surface treatment, todeter adherence of a cell or tissue thereto during the culturingprocess.

Interior walls and side walls may be substantially the same heightrelative to the bottom wall. In some aspects, the interior walls mayhave a height, relative to the bottom wall, that is less than the heightof the side wall, relative to the bottom wall. In such instances,cell-culture medium or other fluid may be added to the container to alevel, h, greater than the height of the interior walls but less thanthe height of the side walls so that the compartments are in fluidcommunication with each other. In certain aspects, one or more of theinterior walls may be coupled to the side walls but not coupled to thebottom wall, leaving a gap between the bottom edge of the one or moreinterior walls and the planar surface of the bottom wall. This gap canbe sized to allow fluid to pass between the compartments of thecontainer without allowing cells, tissues, or 3D scaffolds to passtherebetween. In multi-component embodiments such as illustrated inFIGS. 6A and 6B, the stop 125 may be spaced from the bottom wall 125 sothat, when the components are combined, a gap is left between the bottomedge of the one or more interior walls and the planar surface of thebottom wall. This gap can be sized to allow fluid to pass between thecompartments of the container without allowing cells, tissues, or 3Dscaffolds to pass therebetween. Fluid can be added to a level above orbelow the height of the interior walls. Fluid may be maintained at alevel below the height of the interior walls in order to maintainseparation of 3D scaffolds, cells, and tissues that may float to thesurface of a fluid during culturing.

In certain embodiments, devices of the invention include a lidconfigured to detachably couple to the top edge of the one or more sidewalls in order to enclose the container. The lid can generallycorrespond in shape and size to the bottom wall. In embodiments wherethe one or more side walls taper in or out from the edges of the bottomwall, the lid may be smaller or larger than the bottom wall and cangenerally correspond to the shape and size formed by the top edge of theone or more side walls. The lid may be configured to form an air orwater-tight seal when detachably coupled to the container. In certainaspects, a lid and a container may comprise complementary orinterlocking threads so that the lid may be screwed down onto the upperedge of the at least one side wall. In some embodiments the lid or theupper edge of the at least one side wall may comprise a recessconfigured to accept a gasket wherein the gasket is compressed when thelid is detachably coupled to the container and may thereby form a seal.The gasket may be constructed from any suitable material includingrubber, plastic, metal, nylon, neoprene, or cork. In certain aspects, alid may detachably couple to a container through engagement ofinterlocking tabs on the lid and container. Interlocking tabs mayinclude snap-fit style cantilever prong and recess type connections aswell as screw-type interlocking tabs. FIG. 7 illustrates an exemplarystyle of interlocking tab wherein the lid or the container may include ablock 312 while the other includes a receptacle 310 configured toreceive and lock in the block 312. The interlocking tabs illustrated inFIG. 7 may be engaged by pushing the lid onto the container and thentwisting the lid relative to the container so that the block 312 isinserted down and then into the receptacle 310 as illustrated by thearrow in FIG. 7 .

In certain aspects, the one or more side walls of a container may becoupled to the bottom wall of the container at substantially rightangles and/or the container configured so that the opening at the top ofthe container has an area substantially equal to the area of the bottomwall. An opening may thusly be configured to provide unfettered accessto each compartment within the container from directly above tofacilitate manipulation of materials within each compartment withoutinterference from a tapered or flask-like neck or opening.

In some aspects, a lid may comprise a vent and/or a gas permeablemembrane of a size and shape configured to allow gas to pass into andout of a sealed container while restricting undesirable particles suchas bacteria, endotoxins, and other contaminants. The lid may beconfigured so that, when detachably coupled to the container, the onlyavenue for gas exchange between the interior and exterior of thecontainer is through the filter. The filter may have a variety ofreference pore sizes including 0.2 to 3 gm such as 0.2, 0.5, 1, 1.5, 2,2.5, or 3 gm. The filter may be of a variety of different materials suchas cellulose, nylon, polytetrafluorethylene, polydimethylsiloxane (PDMSsilicone), or glass fiber. Commercially available filter materialsinclude, for example, ePTFE membrane from W. L. Gore & Associates, Inc.,555 Paper Mill Road, Newark, Del. 19711. A filter may also be located ona side wall of the container.

FIGS. 5A and 5B show an exemplary embodiment of a lid 201 according tothe invention. The lid 201 is substantially circular and configured todetachably couple to a substantially cylindrical container such as thecontainer 101 shown in FIG. 1, 2 , or 6A. The lid shown in FIGS. 5A and5B comprises a circular filter 203 placed in the center of the lid. Alid filter may be part of a vent mechanism which includes a cap coveringthe filter material so that gas is vented through the side of the ventand the filter material is protected from fluid splashing. The lid shownin FIGS. 5A and 5B also comprises a downward facing lip with threads 205on the interior surface thereof configured to interact withcomplementary threads on an exterior surface of a curved side wall of acontainer to provide a sealed compression through a screw-typemechanism. FIG. 5B shows a gasket 207, held in a recess of the lid andconfigured to be compressed against the upper edge of a curved side wallof a container. In certain aspects, the top of the lid and/or the bottomof the container may be configured with interlocking tabs and/or spacersto allow for controlled stacking of containers wherein the top containeris spaced above the bottom container so that a filter on the bottomcontainer's lid is still exposed to the exterior atmosphere.

In certain embodiments, a container of the invention may be sized sothat multiple containers may fit on a 7 inch by 14 inch incubator tray.A container may be sized to contain a certain volume of fluid includingat least 1, 10, 50, 100, 250, 500, 750, or 1000 mL. In an exemplaryembodiment, a container 101 of the type illustrated in FIGS. 1 and 2 mayhave an outer diameter of 6 inches while the curved side wall 109 has aheight of 2 inches and the interior walls 107 have a height of 1.5inches and the single, curved interior wall forms a compartment 103 withan inside diameter of 3.5 inches. In an exemplary embodiment of acontainer of the type illustrated in FIG. 3 , the container 101 hasoutside dimensions of 5 inches by 5 inches and the 4 side walls 109 havea height of 2 inches while the interior walls have a height of 1.5inches. The interior walls 107 form one 3.75 inch by 3.75 inchcompartment 103 and 7 1.25 inch by 1.25 inch compartments. In both ofthe above embodiments, two of the specified containers may fit on asingle 7 inch by 14 inch incubator tray.

In certain embodiments, the lid, the container, or components thereofmay comprise a variety of materials. Materials may be biocompatible,endotoxin-free, and/or configured to be sterilized to a variety ofSterility Assurance Levels (SAL) including 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵,or 10⁻⁶, for example. In some embodiments, the lid, container, and/orcomponents thereof may comprise a USP Class N, V, or VI material,including, for example, polypropylene, high-density polyethylene,polystyrene, styrene-acrylonitrile (SAN), polycarbonatestyrene-maleicanhydride (SMA), cyclic olefin copolymers (COC), acrylic,acrylic-polyvinyl chloride alloys, polypropylene copolymers,polysulfone, polymethylpentene, or cellulosic. The lid, the container,or components thereof may comprise a substantially transparent materialto allow visual inspection of the container's contents from outside ofthe container. Containers and lids may be configured as single-usedisposables or may be configured for repeated use and sterilization.Containers and lids can comprise materials which are nonreactive tostandard cell culture media such as EMEM or DMEM.

In some aspects, the interior surface of the bottom wall, the interiorsurface of the one or more side walls, and/or the surfaces of the one ormore interior walls may be smooth; textured with ridges, valleys, pores,or dimples; and/or treated with an agent to deter adherence of culturedcells or tissues and/or the facilitate retrieval from the container byscooping under the cultured tissue with a retrieval tool. In certainaspects, the interior surface of the side walls, interior walls, and/orbottom wall, may be treated with an anti-microbial agent or otherinstrument to prevent microbial growth and/or contamination.

The lid, the container, or components thereof may be produced by avariety of known means including extrusion, injection molding, blowmolding, or rotational molding. The container may be formed from asingle piece of material or from multiple pieces which are subsequentlycoupled together. The lid may be similarly formed. Where constructedfrom multiple pieces, the pieces maybe joined a variety of knowntechniques including welding, or bonding using a bonding agent such as abiocompatible adhesive.

In a preferred embodiment, the lid, container, or components thereof aremade using thermoforming. In thermoforming, a sheet of material, such aspolystyrene is heated until pliable and then formed to a desired shapeusing a mold. Vacuum pressure may be used to conform the material to themold. The material may then be cooled to regain rigidity, and thenremoved from the mold. The resulting product may then be trimmed orotherwise finished to remove excess material or rough edges.

In some aspects, the lid, container, or portions thereof may be formedusing injection molding. In injection molding, a mold block with ahollow cavity in shape of the desired product is coupled to a reservoirthat can inject molten plastic resin is made. The mold is made in twohalves such that a completed part can be removed from one of the halveswithout any portion being impeded by portions of the mold cavity. Themold is placed in a processing machine capable of clamping the twohalves of the mold together. Molten plastic resin is injected into thecavity at high pressure in order to facilitate rapidly filling thin ordistant volumes of the mold.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

The invention claimed is:
 1. A method for culturing a tissue comprising:providing a container comprising a bottom wall coupled to a bottom edgeof at least one side wall, and an at least one interior wall coupled tothe at least one side wall to define at least a first compartment and asecond compartment within the container, wherein the at least oneinterior wall comprises a bottom edge that does not contact the bottomwall, leaving a gap between the bottom wall and the at least oneinterior wall, wherein the gap allows fluid to pass between the firstand second compartments without allowing cells, tissues, or 3D scaffoldsto pass therebetween; providing a lid comprising a gas-permeablemembrane, wherein the lid is configured to be detachably coupled to atop edge of the at least one side wall; introducing a first living cellof a first cell type to the first compartment and a second living cellof a second cell type to the second compartment in the presence of acell culture medium; exposing the container to conditions promoting cellgrowth to produce a first tissue from the first living cell and a secondtissue from the second living cell.
 2. The method of claim 1, whereinthe first cell type or the second cell type is a chondrocyte,mesenchymal stem cell, fibroblast, osteocyte, osteoblast, synoviocyte,induced pluripotent stem cell (iPSC), embryonic stem cell (ESC),lymphocyte, adipocyte, myofibroblast, hepatocyte, islet cell, monocyte,endometrial regenerative cell, or cancer stem cell.
 3. The method ofclaim 2, wherein the first cell type is a chondrocyte.
 4. The method ofclaim 3, wherein the first tissue is neo-cartilage.
 5. The method ofclaim 2, wherein the second cell type is a chondrocyte.
 6. The method ofclaim 5, wherein the second cell type is neo-cartilage.
 7. The method ofclaim 1, wherein the first living cell or the second living cell ispresent on the bottom wall of the container.
 8. The method of claim 1,wherein the exposing step comprises adding a fluid to a level below theheight of the at least one interior wall.
 9. The method of claim 1,further comprising seeding a 3D scaffold with the first living cell andintroducing the 3D scaffold seeded with the first living cell into thefirst compartment.
 10. The method of claim 1, further comprisingpreparing the first tissue for implantation into a human body.
 11. Themethod of claim 1, further comprising performing testing on the secondtissue to verify viability of the first tissue.
 12. The method of claim1, wherein the lid or the container comprises polypropylene,high-density polyethylene, polystyrene, styrene-acrylonitrile (SAN),polycarbonatestyrene-maleic anhydride (SMA), cyclic olefin copolymers(COC), acrylic, acrylic-polyvinyl chloride alloys, polypropylenecopolymers, polysulfone, polymethylpentene, or cellulosic.
 13. Themethod of claim 1, wherein the container comprises a circular bottomwall, a circular lid, and a single, curved sidewall coupled thereto toform a substantially cylindrical shape.
 14. The method of claim 1,wherein the container comprises at least 3 compartments.
 15. The methodof claim 1, wherein the at least one side wall has a first heightrelative to the bottom wall that is substantially equal to a secondheight of the at least one interior wall relative to the bottom wall.16. The method of claim 1, wherein the gas-permeable membrane comprisesa 0.2 micron filter.
 17. The method of claim 1, wherein the container isconfigured to hold at least 500 mL of fluid.